Labdanes and related diterpenoids form a large group of almost 7,000 organic compounds that display a wide range of biological activity and includes a number with significant pharmaceutical activity, particularly medically relevant anticancer, anti-inflammatory, or antimicrobial effects. In addition, potential uses for many of these generally scarce natural products have yet to be fully explored. Biosynthesis of these complex compounds is initiated via a poorly understood acid/base catalyzed (class II) reaction, which forms the bicyclic core structure that defines this super-family of natural products. This bicyclic core is then specifically elaborated by a more typical allylic diphosphate ionization driven (class I) reaction. Interestingly, these mechanistically distinct reactions are mediated by phylogenetically related terpene synthases, albeit in different active sites. Despite the functional importance of the two classes of labdane-related diterpene synthases in initiating the biosynthesis of large numbers of natural products with both realized and potential medical significance, little is known about the enzymatic determinants underlying substrate and product specificity. We propose to investigate these consecutive cyclization reactions as a first step towards our long-term goal of engineering terpenoid natural product biosynthesis for pharmaceutical purposes. On the basis of our previously published and preliminary studies, we hypothesize that the class I active site resides in the C-terminal domain, while the class II active site lies at the interface between two other domains. The structurally defined C-terminal domain associated with class I reactions enables immediate study of the observed specificity via macromolecular modeling directed mutational analysis. To characterize class II cyclization more basic mechanistic enzymology studies are proposed to identify the determinants for catalysis, as well as initial investigations of product specificity. In addition, for both classes of reactions we expect to obtain significant insights through collaborative experimental structure determination, and via continued identification of functionally novel enzymes, along with initial metabolic engineering studies. Thus, the proposed studies will elucidate the structure-function relationships underlying the consecutive class I I/class I cyclization reactions in labdane-related diterpene biosynthesis, and will have a broader impact in further increasing our understanding and use of terpenoid enzymatic cyclization more generally. [unreadable] [unreadable] [unreadable]